Conformational inhibition of the hepatitis C virus internal ribosome entry site RNA - PubMed (original) (raw)
Conformational inhibition of the hepatitis C virus internal ribosome entry site RNA
Jerod Parsons et al. Nat Chem Biol. 2009 Nov.
Abstract
The internal ribosome entry site (IRES), a highly conserved structured element of the hepatitis C virus (HCV) genomic RNA, is an attractive target for antiviral drugs. Here we show that benzimidazole inhibitors of the HCV replicon act by conformational induction of a widened interhelical angle in the IRES subdomain IIa, which facilitates the undocking of subdomain IIb from the ribosome and ultimately leads to inhibition of IRES-driven translation in HCV-infected cells.
Conflict of interest statement
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Figures
Figure 1
The HCV IRES RNA target. (a) Secondary structure of the HCV 5′ NTR (nucleotides 1–341 of HCV genotype 1b) that contains the IRES element. In addition to residues of the NTR, the IRES includes 26 nt of the reading frame in the hairpin loop of domain IV. (b) Secondary structure of the IRES subdomain IIa. Arrows indicate protection from RNase A digestion at an internal loop of IIa in the presence of benzimidazole 1.(c) Three-dimensional structure of the IIa-1 RNA (Supplementary Fig. 1a) corresponding to the subdomain IIa. Mg2+ ions are shown as spheres. (d) Benzimidazole inhibitors of the HCV replicon. Compound 1 has a binding affinity for the IRES subdomain IIa of KD=0.72μM, as determined by mass spectrometry, and inhibits HCV replicon at EC50=5.4μM. Compound 2 is a precursor to 1.
Figure 2
Normalized FRET signal for titrations of Cy3/Cy5-labeled IIa-2 RNA with benzimidazole ligands (a, compound 1; b, precursor 2) in the presence of 2mM Mg2+. Fitting of dose-response curves resulted in EC50 values for ligand binding of 600±80nM for compound 1 and 90±75μM for precursor 2. An interhelical angle of 112±5° was calculated from asymptotic FRET efficiencies for the ligand-bound state of the IIa RNA. Error bars represent ± s.d. calculated from three independent titrations.
Figure 3
HCV translation inhibition by benzimidazole 1 in human Huh-7.5 cells. (a, b) Inhibition of luciferase reporter expressed 4h (a) and 8h (b) after transfection with the BM4-5 FEO HCV replicon RNA. Fitting of dose-response curves results in IC50 values for translation inhibition by compound 1 of 4.0μM (4h) and 9.5μM (8h). (c) Comparison of luciferase reporter expressed 4h after transfection with wild type or A57U IRES mutant RNA in the absence and presence of inhibitor 1. In the control (left, no inhibitor) reporter expression was normalized to wild type. Expression in the presence of 25 μM inhibitor (right) was normalized to control for each wild type and mutant. While the A57U change originates from an HCV clinical isolate, the efficiency of IRES-driven translation is drastically reduced in this mutant (15% of wild type activity). (d) Inhibition of luciferase reporter expression after 48h in cells stably transfected with HCV replicon. Fitting of a dose-response curve results in an IC50 value for replicon inhibition by compound 1 of 2.0μM. Assessment of cell viability in the presence of compound ruled out that signal decrease was due to cytotoxicity (Supplementary Fig 8). Error bars represent ± s.d. calculated from triplicate experiments.
Figure 4
References
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